We previously studied spirochetes (B. burgdorferi) that have converted from spirochetes to cystic forms in CSF in vitro using the same methods as mentioned above [11]. With all these methods used in this study (TEM, AO, DF), the cystic structures observed in the CSF of the MS patients are morphologically similar to cystic forms of spirochetes. We found that cysts which are produced by inoculating B. burgdorferi in CSF at 37 C can be PCR negative using conventional DNA extraction and OspA primers (unpublished observation). This is either because the cyst wall inhibits the entrance to the genome or because the genomes of spirochetes have been changed. We have also to keep in mind that PCR detection of B. burgdorferi spirochetes often may give false-negative results [19).

The positive IgG index associated with MS in our patient cohort proves that the patients had an active inflammatory process in the CNS (Table 1). Inflammatory processes in the b~ and spinal cord of virtually any cause are usually less intense than inflammation in peripheral tissues and some microbiological agents, including spirochetes, provoke a very gentle inflammatory response [20, 21 ]. Considering the nature of MS, this disease could very well be a chronic infection and the clinical picture of MS has repeatedly been confused with neuroborreliosis [22-26). Therefore, we have both microbiological and some clinical support for the hypothesis that the cystic structures found in the CSF of the MS patients may originate from spirochetes which could be the causative agents of MS.

2... the frequency of relapses may be highly dependent on the chosen treatment and the phase of the disease [23−26]. A study of cytomorphic variations of B. burgdorferi isolates from patients with or without antibiotic treatment showed that penicillin can induce membrane-derived vesicles (cysts or spheroblast L-forms) in vivo [27]. This conversion of mobile Borrelia to cystic forms was subsequently observed for ceftriaxone, doxycyclin [17], ciprofloxacin [18] and vancomycin [12] at concentrations achievable in vivo. The fact that B. burgdorferi has the ability to convert (and reconvert) to cystic forms both in vivo and in vitro [1,4−6,10,14,15,21,27,28] may be regarded as an explanation why the infection may be persistent and reactivate. Therefore, it is reasonable to suggest that all germinative forms of the bacterium (and not only the motile form) should be destroyed so that Lyme borreliosis can be treated effectively.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole. INTERNATIONAL MICROBIOLOGY (2004) 7:139?142.

3. Concurrent Neocortical Borreliosis and Alzheimer's Disease Demonstration of a Spirochetal Cyst Form

Annals of the New York Academy of Sciences 1988, 468-470

Alan B. MacDonald, Southampton Hospital, Southampton, NY 11968

A 71 year old man died in Arizona 3 years after the onset of progressive dementia. A diagnosis of probable Alzheimer's disease was based on clinical criteria. The brain was removed at autopsy, frozen (unfixed), and transported to the Department of Pathology, University of California, San Diego, School of Medicine where it was stored at -70 degrees C for further study. The author received the frozen brain and utilized methods previously described (1) for in vitro culture, cytologic, immunohistochemical , and silver impregnation studies. Argyrophilic plaques and neurofibrillary tangles were found in the frontal lobe and hippocampal formation is sufficient number to establish the neuropathologic diagnosis of Alzheimer?s (fig 1 A). Spirochetes were visualized in imprint preparations of freshly thawed frontal lobe cortex with monoclonal antibody H5332, which specifically binds to the outer surface membrane of Borrelia burdorferi (fig.2). Borrelia spirochetes were recovered from cultures of freshly thawed cerebral cortex and hippocampus in Barbour-Stoenner-Kelly medium. An unexpected observation was the identification of cystic forms of the Borrelia spirochete in dark-field preparations of cultured hippocampus, and in imprints of hippocampus using the monoclonal antibody H9724, which binds to class-specific axial filament proteins of Borrelia spirochetes. Oil immersion examination of sections from the hippocampus impregnated with silver disclosed a rare cystic structure (fig. 1B). Previous workers have identified spirochetal cyst forms in cultures of non-pathogenic treponemal spirochetes and have suggested that spirochetes have a complex life cycle. (2-5) Dark-field examination of aged cultures of the B31 reference strain of Borrelia burgdorferi disclosed cystic structures similar to the cysts found in the autopsy brain culture.

The following hypothesis is offered based on these observations. Borrelia spirochetes have a complex life cycle which includes corkscrew shaped forms, uncoiled filamentous forms, L-forms lacking a cell wall, cystic and ameboid forms, and granular forms. These forms may exist as either extracellular or intracellular pathogens. The cystic form of Borrelia burgdorferi may explain the Pick body, which is found in Pick?s disease, and the granular form of Borrelia may explain granulovascular degeneration of nerve cells in the hippocampal formation in Alzheimer?s disease. A cystic form of the Borrelia spirochete would explain the ability of the microbe to persist in the host during a prolonged periodof asymptomatic clinical latency, which spans the period between primary infection and the expression of tertiary manifestations of neuroborreliosis.

Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cystinoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

ABSTRACT:
The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive structures represent various forms of B. burgdorferi rather than staining artifacts. These "atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this bacterium.

ABSTRACT:
As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated from antibiotically treated and untreated patients with the clinical diagnosis of definite and probable Lyme borreliosis and from patient specimens contaminated with bacteria were investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron microscopy revealed diverse alterations. Persisters isolated from a great number of patients (60-80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation. Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells to replicate, the long period of adaptation to growth in MKP-medium and the mycoplasma-like colonies after growth in solid medium (PMR agar) suggest that B. burgdorferi produce spheroplast- L-form variants. With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion.

5. ...For example, B. burgdorferi has the ability to survive in divergent conditions of mammals and ticks by existing in a variety of forms that are ultrastructurally and metabolically distinct. Even in the tick, altered morphologic forms of B. burgdorferi are present [43], but in the mammal, selective pressure from mammalian immune surveillance results in these altered forms becoming more common. These "host adapted" forms generally display altered morphology to varying degrees and are referred to collectively as L-forms or spheroplasts. B. burgdorferi spheroplasts, of which cystic forms and granules are sub-types, have been extensively documented in vitro and in vivo [44?53], both extracellularly and intracellularly [27, 47, 54?57]. Their ability to revert from host-adapted forms back to helical forms under appropriate conditions has been demonstrated in vitro [47, 58, 59].

To the uninitiated, it may be tempting to infer that B. burgdorferi cystic forms are degenerative bacterial fragments. This is not the case, since researchers have demonstrated protein synthesis requirements for spirochetal conversion into the spheroplast form [44]. Indeed, it has been unequivocally proven that B. burgdorferi cystic forms are virulent and infectious. Their infectivity, survival under extreme environmental conditions, and ability to revert back to helical forms in vivo have all been demonstrated by inoculation of B. burgdorferi cysts into mice and subsequent recovery of helical spirochetes from the animals [60]. As such, host-adapted forms of B. burgdorferi are considered to be major factors in the relapsing and persistent nature of Lyme disease [61?63].

Just as B. burgdorferi spheroplasts have altered metabolic requirements for growth, so too, do they have unique antibiotic sensitivities, altered surface protein expression, dramatically reduced surface area presented for immune surveillance, and the ability to cause multiple potential problems for PCR analysis. All of the foregoing helps to explain observations of antibiotic resistance, seronegativity, and even frequent PCR negativity in active disease [51, 54, 59, 63, 64]. The failure to address the complexities of the borrelial life cycle in the work by Klempner et al. is a serious error. For example, the fact that cystic forms demonstrate sensitivity to metronidazole while their helical kin are resistant, illustrates the point that B. burgdorferi spheroplasts have altered antibiotic resistance [65]. Attention to these forms during the initial study design might have resulted in different treatment decisions, with consideration that a cell wall-attacking cephalosporin may not have been the ideal antibiotic choice for treatment of cell wall-deficient organisms in patients with late-stage Lyme disease.

In addition, had the authors addressed the intracellularity of B. burgdorferi, this might have broadened their choices of antibiotic therapy. Although the utility of ceftriaxone for Lyme disease has been documented, it has been similarly documented that this agent frequently does not fully eradicate human B. burgdorferi infections [19]. Cephalosporins do not achieve intracellular penetration, a fact that may partially explain well-known treatment failures associated with late stage Lyme disease. Indeed, B. burgdorferi has been documented within a variety of cell types, including but not limited to endothelium, fibroblasts, lymphocytes, macrophages, keratinocytes and synovial cells [17, 51, 54, 66?70]. These findings are critically important since chronic infections are highly dependent on intracellular asylum as a mode of persistence, and localization within eukaryotic cells protects B. burgdorferi from antibiotics [71, 72]. It is particularly surprising that the lead author agreed to use ceftriaxone in this study, since he previously authored a paper on the fibroblast-mediated protection of B. burgdorferi in vitro from concentrations of ceftriaxone achieved in vivo for the treatment of Lyme disease [71]...

..Indeed, there have been a number of peer reviewed publications demonstrating persistent infection with B. burgdorferi in humans despite multiple and extended courses of antibiotic therapy [19]. Persistent infection has been demonstrated repeatedly by both polymerase chain reaction (PCR) and histopathology [20-23]. Chronic infection has also been demonstrated by culture despite the well-known difficulties in harvesting B. burgdorferi from Lyme patients, and culture positivity has even been found in patients who are seronegative for the Lyme spirochete [24?30]. In light of such data, it would be illogical to assume that persistent symptoms in chronically ill Lyme disease patients are not related to active infection with B. burgdorferi. Ironically, and in direct opposition to the extensive body of published data, some researchers have attributed chronic symptoms compatible with Lyme disease to alternative vague diagnoses, such as "post-Lyme syndrome," fibromyalgia, or chronic fatigue syndrome [31,32]...

Here is proposed a hypothesis that a completely unsuspected biology exists for pathogenic spirochetes, namely that the cystic spirochetal forms (long thought to be static and resting or just a dormant cohort) actually are capable of killing mammalian host cells. At least two "lethal" scenarios are proposed; first, the host cell destruction from the "inside out" by small caliber cystic forms invading the host cell cytoplasm, and second host cell destruction by engulfment of entire host cells by large caliber cystic spirochetal forms. Conventional thinking about spirochetal cyst forms is divided between two polar spheres of influence; one a majority community that completely denies the existence of spirochetal cyst forms, and a second group of academically persecuted individuals who accepts the precepts of such antebellum scientists as Schaudinn, Hoffman, Dutton, Levaditi, Balfour, Fantham, Noguchi, McDonough, Hindle, Steiner, Ingraham, Coutts, Hampp, Warthin, Ovcinnikov, and Delamater. Microscopic images of cystic spirochetes are difficult to ignore, but as has been the case in this century, academic "endowments" have nearly expunged all cystic spirochetal image data from the current textbook versions of what is the truth about the spirochetaceae. If the image database from the last century is obliterated; many opportunities to diagnose will be lost. Variously sized cystic spirochetal profiles within diseased nerve cells explain the following structures: Lewy body of Parkinson's disease, Pick body, ALS spherical body, Alzheimer plaque.

Borrelia infection is therefore a unifying concept to explain diverse neurodegenerative diseases, based not entirely on a corkscrew shaped profile in diseased tissue, but based on small, medium and large caliber rounded cystic profiles derived from pathogenic spirochetes which are..."hiding in plain sight".

Igor Gruntar,1 Tadej Malovrh,2 Rossella Murgia3 and Marina Cinco3
Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse
environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

Cystic Form of Borrelia: Susceptibility to Treatment with Metronidazole

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole.

Brorson O; Brorson S. 1999.

Methodology for Generation of Cystic Forms of Borrelia burgdorferi

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.

Brorson O; Brorson S. 1998.

Transformation of Borrelia Spirochetes to Cystic Forms in Spinal Fluid

In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium.

"The form of spirochetes was unusual, i.e. cyst-like (Fig.5). The surface membrane of cyst of Bb was antigenically different, negative with McAb H 5332 , positive with lectin WGA in the IEM method (Fig.7)
in contrast to the material of the spirochetes inside the cyst, which was positive with McAbH 5332 in the outer envelope on cross section

In Vivo Findings: Observation of Borrelia Cysts in Autopsy of Human Brain

Hamilton Montana, Conventional corkscrew and Concurrent Cystic forms, 1000x magnification, Oligreen stain (Invitrogen), Alan MacDonald, MD, unpublished image, 2006, Note: These cystic forms are NOT detected in conventional Acridine orange or in Picogreen (Invitrogen ) stains for double strand spirochetal DNA, and therefore would be "invisible" with standard detection modalities. These profiles overturn the concept that only during times of stress will you expect to find Cysts of Borrelia. These cyst forms developed in BSK culture medium during log phase normal growth of the reference strain of Borrelia burgdorferi. http://66.102.9.104/search?q=cache:i5Z7 ... cd=7&gl=fi

-------------------------------------------

A Proposal for the Reliable Culture of Borrelia burgdorferi from Patients with Chronic Lyme Disease, Even from Those Previously Aggressively Treated

....." RESULTS
Of the 47 patients with chronic Lyme disease, 43 (91%) cultured positive for B. burgdorferi. while 23/23 (100%) of
the controls cultured negative. Many of the cultures were clearly spirochetes when examined under light microscopy (Figures 1-3). Immuno-electron microscopy and Osp A PCR confirmation provided additional confirmatory evidence as to the identity of the spirochetes (Figures 4-7). The slide cultures consistently demonstrated the fastest and most abundant yields. With this technique, placement in the Coplin jar allows for varying gradations of oxygen tension. Sometimes spirochetal growth can be seen after as little as 20 h. appearing as a band near the upper end of the smear.

DISCUSSION
An attempt to culture B. burgdorferi from the blood of previously aggressively treated chronic Lyme disease patients seemed at first a monumental task. Before undertaking this effort, we therefore had to be as sure as possible that the organisms were indeed present in the blood of these patients. As a first step, we scrutinized a report where B. burgdorferi had been cultured from the blood of patients with early untreated disease. From this group of patients it had been noted in follow-up that subsequent blood cultures became routinely negative after antibiotic therapy, despite 71% of the patients remaining symptomatic [9]. Three possibilities readily come to mind for the explanation of this paradox: either 1) the infection is cleared, but a post-infectious process continues, or 2) the organism is cleared from the blood rapidly. but finds a pathogenic harbor elsewhere, or 3) the organism is maintained in the blood in an altered state which cannot be cultured on routine media.

In response to the first possibility, the notion of a post-Lvme syndrome has countless flaws. A post-infectious syndrome could not explain the observation that patients with "post-Lyme" or "post-Lyme fibromyalgia" responded to re-treatment with antibiotics, only to relapse with its discontinuation [13-15]. With the advent of PCR, antigen capture, and the benefit of those rare successful culture experiments even in the face of prior "curative treatment" [3-8]. the notion of "post-Lyme" should have been dismissed long ago. In response to the second possibility, given the common finding of circulating immune complexes with Lyme disease, we thought this unlikely [16]. Thus we were left with the third and most logical possibility. Specifically, we chose to pursue the organism in its cell wall-deficient state, i.e. L-forms, as previously reported [17].

Although L-forms will complex with fluorescent antibody to B. burgdorferi, only as they revert to classic parent forms can the typical spirochetal morphology be seen. There has been a considerable spectrum of cell wall deficiency demonstrated in our laboratory. B. burgdorferi may exist in various forms depending on its environment. In addition to the spirochetal form, we have demonstrated its growth both as amorphous L-forms and rounded giant L-bodies which have been previously described as cystic forms [11, 18]. As B. burgdorferi reverts from cell wall deficiency with the rebuilding of its cell wall, classic spirochetal forms can be seen. Most often, in our cultures, B. burgdorferi can be seen in varying stages of reversion, i.e. some L-dependent spirochetal forms within an L-form colony.

The L-form variants, osmotically fragile by nature, require precise conditions to grow in culture. our medium and methodology are specifically designed for the fostering of cell wall-deficient organisms and their reversion to classic parent forms. In most instances, the methods must be followed precisely. Even small variations produce no growth. For example, 2% yeast extract instead of 1% is inhibitory. or if the yeast extract is autoclaved with the rest of the medium instead of separately. that too will be inhibitory. However, there is one aspect of B. burgdorferi's growth characteristics which we found to be remarkably non-fastidious. The organism can be easily grown throughout a wide range of pH, from 6.8-7.8. This explains the different ratios of NaHC03 used in the various types of culture mediums. We are still not sure about the optimal pH for culture. Future research will address that question more specifically.

It should be noted from this study that currently accepted standards for serologic diagnosis seem to be inadequate. Only a small minority of participants in the study had positive Lyme ELISAs. Under the current recommendations for two-tier testing by the CDC/ASTPHLD, 91% of the patients in the study would have been misdiagnosed as not having Lyme borreliosis.

It is hoped that our work will help to end a medical controversy which has been going on for far too long. This study proves that chronic Lyme disease is of chronic infectious etiology, and that even antibiotic treatment well in excess of current recommendations is not necessarily curative. Given the flaws in currently accepted serologic diagnostic criteria, it is likely that Lyme borreliosis is vastly underdiagnosed. May this research help to focus the scientific community on effective curative therapies for patients with chronic Lyme disease.

It should also be noted that, in addition to its utility in growing B. burgdorferi, the MPM medium may be useful for culturing a variety of other spirochetes from patients."

Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

It has been demonstrated recently that cells of Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease, transform from mobile spirochetes into nonmotile cystic forms in the presence of certain unfavourable conditions, and that cystic forms are able to reconvert to vegetative spirochetes in vitro and in vivo. The purpose of this study was to investigate the kinetics of conversion of borreliae to cysts in different stress conditions such as starvation media or the presence of different antibiotics. Using the same experimental conditions we also investigated the possible role in cyst formation of RpoS, an alternative sigma factor that controls a regulon in response to starvation and transition to stationary phase. We observed that beta-lactams penicillin G and ceftriaxone, the antibiotics of choice in Lyme borreliosis treatment, favoured the production of cysts when used with serum-depleted BSK medium. In contrast, we observed a low level of cyst formation in the presence of macrolides and tetracyclines. In order to elucidate the role of the rpoS gene in cyst formation we analyzed the reaction of the rpoS mutant strain in comparison with its wild-type in different conditions. Under the same stimuli, both the wild-type borrelia and the rpoS knock-out isogenic strain produced cystic forms with similar kinetics, thus excluding the participation of the gene in this phenomenon.

Our findings suggest that cyst formation is mainly due to a physical-chemical rearrangement of the outer membrane of Borrelia burgdorferi sensu lato leading to membrane fusion and controlled by different regulation mechanisms.

Background
The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis. Method: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes.

Results: Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods.

The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex.

Conclusion: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.

...The persistence of more resistant atypical cystic and granular forms of Treponema pallidum, which are less sensitive to chemicals and antibiotics, are responsible for the long latent stage in chronic syphilis and for the infectivity of tissues devoid of the demonstrable vegetative form of spirochetes. The intracellular localization of Treponema pallidum is another way of evading from destruction by the host immune system [30, 39]. Virtually all types of mammalian cells can be invaded by Treponema pallidum resulting ultimately in functional cell damage and cell destruction.

Recently we reported evidence that Borrelia burgdorferi can also persist in the brain in chronic Lyme neuroborreliosis and, in analogy to Treponema pallidum, may cause dementia, cortical atrophy and amyloid deposition [3, 49, 51]. Only limited data have previously been available on the presence of atypical, cystic forms of spirochetes in the brain in chronic Lyme neuroborreliosis. Whether such forms may eventually cause functional damage and cell death is still not certain.

Here we analyzed atypical, cystic forms of Borrelia burgdorferi induced by unfavorable culture conditions and compared these with forms observed following 1 week of infection of primary chicken and rat neurons, as well as primary rat and human astrocytes. We also analyzed whether similar atypical and cystic forms may occur in vivo in brains of patients with pathologically and serologically confirmed Lyme neuroborreliosis and compared them to the atypical forms of Treponema pallidum in brains of patients with general paresis. The results show that under harmful culture conditions, the typical forms of Borrelia spirochetes are replaced by atypical forms varying from ring-shaped and cystic forms to fine single granules of almost submicroscopic size. These results are in harmony with previous observations [8, 55, 66]. The effect of osmotic shock induced with cold distilled water or heat shock was identical to those previously observed in other spirochetes [25, 67] Thioflavine S and Congo red had a similar effect. The mechanism of the harmful effect of these dyes is not known. They may act by binding to the outer sheath of Borrelia spirochetes [e.g. 56]. Thioflavin S and Congo red are widely used to detect amyloid deposits in affected tissues. Several observations suggested that Borrelia burgdorferi possesses amyloidogenic proteins [51, 68, 69]. Peptides derived from the OspA single-layer beta-sheet showed fibrillary amyloid formation, which may be an explanation of the binding of Thioflavin S and Congo red to the outer surface of Borrelia burgdorferi.

Atomic force microscopy (AFM) analysis showed rolled Borrelia spirochetes inside of a cyst covered by a thin outer membrane. This has also been observed in various types of spirochetes [e.g. 11, 38, 70] including Borrelia burgdorferi [8] by transmission electron microscopy analyses. Uni- or multi-spirochetal cysts may be formed. We illustrated by atomic force microscopy (AFM) rolling of two Borrelia spirochetes to form a cyst. The size of such cysts depends on the number of spirochetes packed inside of the cyst [8]. We observed bleb formation, connected to Borrelia spirochetes by a fine stalk, in both Borrelia strains. Thin newly formed spirochetes attached to spirochete cells, and to free minute granules were also observed.

Similar atypical, cystic and granular forms were observed in primary neuronal and astrocytic cell cultures exposed for 1 week to the Borrelia burgdorferi strains B31 and ADB1. ...
_________________________________________________________________

Background
The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis. Method: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes. Results: Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex. Conclusion: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.

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Antibacterial soap, hand sanitizer and antibiotics are all substances that we use in an attempt to kill bacteria that might make us sick.Whether we are concerned about getting strep throat, bacterial meningitis or something else, these prevention methods can offer protection.

However, some bacteria, such as those that cause Staph and MRSA infections, are becoming increasingly resistant to antibiotics. Since the 1930s, researchers have been aware that bacteria may be able to resist treatment because they can morph into the L-form, or bacteria lacking cell walls.

Until the 1980s, not much else could be known about the L-form, but now, researchers at the Bloomberg School of Public Health have used a wide variety of modern molecular tools to learn more about the origin and biological functions of the L-form bacteria.

Ying Zhang, a professor of molecular microbiology and immunology at Bloomberg, is the senior author of the study, which was published in PLoS ONE last month.

Not all bacteria can transform into the L-form, but those that can include Bacillus anthracis (anthrax), Treponema pallidum (syphilis), Mycobacterium tuberculosis (tuberculosis), Heliobacter pylori (stomach ulcers and cancer), Borrelia burgdorferi (Lyme disease) and Escherichia coli (food poisoning). Zhang's team used E. coli to create a culture of L-form bacteria.

Although it had been difficult to culture L-form bacteria before, Zhang and his team created a new method that more closely simulated the in vivo conditions in which these bacteria form.

"The presence of antibiotic stress is cell wall inhibiting, like penicillin," Zhang said. To prevent the cells from bursting because of this increased stress, Zhang's team added sucrose to the cell media.
This culture represented the mechanism that occurs in the body. "L forms are formed in response to stress," Zhang said. "They have a different mode of survival and replication from classical bacteria." The cell wall-deficient bacteria cluster together in the shape of a fried egg rather than the smooth, homogeneous appearance of wild-type bacteria cultures.

Not only are L-form bacteria difficult to culture and therefore study, but this "fried egg" cluster is part of what makes the L-form bacteria resistant to antibiotics, in addition to the fact that they do not have cell walls for commonly used antibiotics to disintegrate.

Once Zhang and his team were able to successfully culture L-form E. coli, they screened for and identified mutants that fail to grow at the L-form. From these mutants, they were able to discover a series of genes that were linked with the inability to grow in the L-form.

"These fall into four to five different categories involving extracellular matrix synthesis, membrane proteins, membrane biogenesis, DNA repair as well as iron metabolism and energy metabolism," Zhang said.

Their identification of these genes and their effect on L-form bacterial expression is a resounding discovery because it was impossible to do before, what with the difficulty of culturing the L-forms of various bacteria. Zhang noted, however, that although his team managed to create and study a culture of L-form bacteria, their study cannot be universal.

"What we can culture is only a small percentage - probably less than 1 percent - of all bacteria on earth," Zhang said.
"They exist in nature and grow easily, but we're limited to what we can grow and the form of bacteria that can grow. Bacteria can grow a variety of different forms even for the same species, and can change forms under different conditions. L-forms are one example of changing under antibiotic stress."

These L-forms of various bacteria may be the underlying reason for chronic resistant and recurring diseases, such as sarcoidosis, various forms of inflammatory bowel diseases and rheumatoid arthritis. Zhang is confident that there will be many practical applications of this discovery.

"It is possible, with our discovery of the L-form genes to develop new antibiotics and more effective ones that can be used with current ones as well as new vaccines to . . . allow these forms to be eliminated by the immune system," he said.

The article below (by Steve Nery) was published in at least three Maryland newspapers last week- the Star Democrat (front page), the Kent County News and the Record Observer.

The Kent County News

Lyme disease victims eye beneficial study.

By Steve Nery

BALTIMORE Lyme disease patient advocates are hopeful that new studies on especially resistant forms of bacteria at Johns Hopkins could help lead to less restrictive treatment guidelines.

The November issue of the Johns Hopkins newsletter contains an article about research at the Bloomberg School of Public Health on bacteria that can morph into the "L-form" bacteria that lack cell walls that are resistant to antibiotics. They include Borrelia burgdorferi, the bacteria that causes Lyme disease, as well as the bacteria responsible for anthrax, syphilis, tuberculosis, food poisoning and stomach ulcers.

The Infectious Diseases Society of America recommends the use of an antibiotic for a maximum of four weeks to treat Lyme disease, but many patients, especially those not diagnosed swiftly following a tick bite, report effects that can continue to linger for life. Droves of Lyme disease patients spend thousands in out-of-pocket dollars to receive treatment from the few doctors that don't follow the IDSA guidelines.

Ying Zhang, Yanking Yang and William A. Glover authored a study published last month in PloS ONE. The researchers used modern molecular tools to learn more about the origin and biological functions of the L-form bacteria. Researchers have known about the existence of this form of bacteria since the 1930's, but little beyond that.

Zhang, a professor of molecular microbiology and immunology at Bloomberg and the senior author of the study, said in the newsletter that the bacteria take on the L form in response to stress caused by antibiotics. The wall-less cells cluster together in the shape of a fried egg, which makes them ever more resistant to antibiotics.

The team successfully cultured E. coli L-form bacteria and discovered genes that were linked with the inability to grow in the L-form.

These L-forms of various bacteria may be the underlying reason for chronic diseases such as rheumatoid arthritis, according to the newsletter.

"It is possible, with our discovery of the L-form genes, to develop new antibiotics and more effective ones that can be used with current ones as well as new vaccines to ? allow these forms to be eliminated by the immune system", Zhang said in the publication.

Patients with chronic Lyme disease hope new research will change the Infectious Diseases Society of America's Lyme disease treatment guidelines, which are under review as ordered by Connecticut Attorney General Richard Blumenthal.

"Hopkins proving L-form bacteria is not killed by antibiotics while in this form shows that the chronic and debilitating symptoms, disability and death can all be related to ongoing infection in those that have been exposed", said Lucy Barnes, director of the Lyme Disease Education and Support Groups of Maryland

Margulis and colleagues spirochete research may point to new Lyme treatment

[] In Proceedings of the National Academy of Sciences, Distinguished Professor Lynn Margulis, Geosciences, UMass Amherst advanced and former students James MacAllister and Andrew Wier and their colleagues in Norway and Canada, report new details about the life history of the spirochete bacterium, Borelia burgdorferi, which causes Lyme disease. Their study leads them to suggest that clinical trials of the antibiotic Tigecycline are warranted. This drug potentially provides a treatment regimen for people with Lyme disease that has worked, so far only in the laboratory, to destroy spirochetes, they point out.

The study represents a lifetime of work by medical microbiologist Oystein Brorson and his pathologist cousin, Sverre-Henning Brorson of Oslo, Norway, says Margulis, a winner of the Darwin and Wallace Medal from the Linnean Society of London. She recently returned to campus from a year as Eastman Professor at Balliol College, Oxford University.

B. burgdorferi spirochetes take up residence in tissues of people bitten by ticks that carry the bacteria in their guts and inject them via saliva. The spirochetes most familiar form is an agile, spiral-shaped swimmer associated with acute symptoms of Lyme disease and rapid reproduction of the bacteria. But as Margulis explains, these bacteria can go underground and persist for years by entering a self-protective, quiescent stage known as a spirochete round-body (RB) propagule. In this state, they better resist what scientists call unfavorable environmental conditions such as starvation, desiccation and exposure to antibiotics such as penicillin and deoxycillin. Chronic Lyme disease symptoms correlate with the continuous presence of reversible RB propagules in patients moist tissues.

What the Brorsons work shows is that, unlike other antibiotics, Tygecycline administered at the correct dosage and timing destroys the bacterium even when it has protected itself in this quiescent stage. Other antibiotics, if they do anything at all, simply cause B. burgdorferi to enter its RB propagule state and wait out the treatment. â??Tigecycline is, so far, the only known antibiotic that destroys the Lyme disease spirochete in both the growing and the quiescent RB stages of its life historyâ? Margulis notes.

She and her students are interested in spirochetes, in particular two individuals found embedded in ancient (15 million-year-old) amber and their RB propagules, because of the Serial Endosymbiosis Theory of the evolution of nucleated cells. It posits that animal, plant, fungal and other cells with nuclei evolved by symbiotic merger of two types of bacteria, at least: A spirochete (belonging to the eubacterium group) that reversibly develops RB propagules in response to changing conditions, and Thermoplasma, from the archaebacterium group, a sulfide-gas-making microbe that lives in nearly boiling and very acidic waters (more acid than our stomachs). In their PNAS paper, the authors state that chronic spirochete infections in humans when seen in their ecological-evolutionary context are examples of symbioses that have evolved over geologic time.

Image: Persistent quiescent Lyme disease spirochete (Borrelia burgdorferi) round bodies (RBs) may endure for years. They stain red as seen with here with a fluorescence microscope (scale bar = 8 micrometers). One RB is seen at very high magnification with an electron microscope (EM) thin section in the lower left inset). After incubation in a favorable supportive blood serum growth medium for over one month the RBs develop into normal helical swimmer spirochetes as seen in the EM photo of the upper right inset (bar = 1 micrometer). (Photo courtesy of Oystein and Sverre-Henning Brorson)
February 17, 2010.

The aim of the present paper was to evaluate cyst formation and growth parameters of Borrelia garinii in a range of media differing in formulation and cost. A qualitative assessment of morphology and motility of B. garinii was conducted. All media were prepared aseptically and used in test tubes or Petri dishes. For each medium, the initial spirochete concentration was standardized to 103 spirochets/mL. The following culture media were suitable to grow B. garinii: Barbour-Stoenner-Kelly, brain heart infusion and PMR. Growth was minimal at six weeks post-inoculation and maximum spirochete density was observed between 9-12 weeks.

Often, the cultures developed cysts of different sizes, isolated or in groups, with a spiraled portion of variable sizes, mainly in unfavorable culture media.

Brazilian Lyme disease-like illness, also known as Baggio-Yoshinari syndrome (BYS), is a new and interesting emerging tick-borne disease, caused by Borrelia burgdorferi sensu lato spirochetes, only during its cystic forms.

It has been assumed that the peculiar clinical and laboratory features of BYS are consequential to the absence of a human sucker Ixodes ricinus complex tick at risk areas in Brazil, supporting the concept that the borrelia phenotypic expression pattern is modified as it is transmitted through the host.

Borrelia burgdorferi, the agent of Lyme borreliosis, has the ability to undergo morphological transformation from a motile spirochetal to non-motile spherical shape when it encounters unfavorable conditions. However, little information is available on the mechanism that enables the bacterium to change its shape and whether major components of the cells ? nucleic acids, proteins, lipids ? are possibly modified during the process.

Deducing from investigations utilizing electron microscopy, it seems that shape alteration begins with membrane budding followed by folding of the protoplasmatic cylinder inside the outer surface membrane. Scanning electron microscopy confirmed that a deficiency in producing functioning periplasmic flagella did not hinder sphere formation. Further, it was shown that the spirochetes' and spheres' lipid compositions were indistinguishable. Neither phosphatidylcholine nor phosphatidylglycerol were altered by the structural transformation. In addition, no changes in differential protein expression were detected during this process. However, minimal degradation of RNA and a reduced antigen-antibody binding activity were observed with advanced age of the spheres.

B. burgdorferi spirochetes converted to a cystic form when placed in a deficient culture media. These cysts reverted to motile spirochetes when transferred to growth media.[From the abstract:] "Cyst opening and recovery of spiral-shaped non-motile organisms was induced within 1 min by the addition of either BSKrs+ or rabbit serum (6%v/v, final concentration). Cells regained motility with additional incubation in BSKrs+."

APMIS, 1999;106(12):1131-1141

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.Brorson, O., & Brorson, S.Motile B. burgdorferi spirochetes converted to cysts within 1 minute when placed in distilled water. The cysts reverted to spirochetes after transfer to a growth medium.

APMIS, 1999;107(6):566-576.

(R) Brorson, O., & BrorsonAn in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole.B. burgdorferi cysts were degraded upon incubation with metronidazole (in vitro).

12th International Conference on Lyme Disease and Other Spirochetal and Tick-Borne Disorders., April 8-9, 1999Keynote Address - The Complexity of Vector-borne Spirochetes.Burgdorfer, W."This relatively large Borrelia [Borrelia burgdorferi] is not readily detectable in blood smears or thick drops of Lyme disease patients and susceptible host animals, yet engorgement on infected hosts results in up to 100% infected ticks.... RML [NIH's Rocky Mountain Lab] scientists Dave Dorward and Claude Garon using silver staining, transmission and scanning electron microscopy investigated the nature of naturally elaborated membrane blebs on the surface of cultured B. burgdorferi or free in the medium, and found both linear and circular DNA (Fig.13)... These most recent findings [of RML researchers and others] do confirm the development of membrane-derived cysts, blebs, spherules, vesicles and the potential transformation to motile, helical spirochetes...as a "survival mechanism" of spirochetes to overcome or escape unfavorable conditions." [Willy Burgdorfer, Ph.D., of the National Institutes of Health, is the discoverer of Borrelia burgdorferi.]

Infection, 1998;26(3):144-50

(R) In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H mediumBrorson, O., & Brorson S.. B. burgdorferi transformed into cysts (spheroplast L-forms) within 1-24h of inoculation into spinal fluid. When transferred to a growth medium, the cysts converted back to normal spirochetes after 9-17 days of incubation. The authors suggest that encystment of Bb may explain why cultivation of spinal fluid may be negative in patients with neuroborreliosis. The cysts may be recognized by microscopy.

American Journal of Dermatopathology,1996;18(6):571-9.

(R) Heterogeneity of Borrelia burgdorferi in the skin.Aberer, E., Kersten, et alEncysted B. burgdorferi were found in a skin biopsy. The authors conclude that these are in vivo variant forms of Bb and not staining artifacts. Includes remarkable color videomicroscopy photographs as supporting evidence.

[Diagnosis:] "Borrelia may escape immune surveillance by colony formation and masking within collagen, resulting in seronegativity."

Antimicrobial Agents & Chemotherapy, 1995;39(5):1127-33.(R) Effects of penicillin, ceftriaxone, and doxycycline on the morphology of BorreliaKersten, A, Poitschek C, Rauch S, Aberer, EB. burgdorferi cultures gradually developed cysts and blebs [granules] when incubated in antibiotics. The degree of alteration was strongly correlated with dose and duration.The alterations occured most rapidly with ceftriaxone, then with penicillin; doxycycline caused a smaller percentage of organisms to undergo morphologic changes. Those organisms that remained in typical spirochetal form in the doxycycline cultures showed decreased motility. The authors suggest that these morphologic changes may shed light on the ability of B. burgdorferi to survive

Infection, 1994 ;22(6):401-406.

Ultrastructure of Borrelia burgdorferi after exposure to benzylpenicillinSchaller, M. et al&B. burgdorferi were observed to form cysts and blebs when treated with penicillin G. "These structures were not found under optimal culture conditions. One may speculate that the borreliae could escape the action of the antibiotic by developing such spherical bodies." (p. 404)

Abstracts of the V International Conference of Lyme Borreliosis, Arlington, VA.1992

Borrelia burgdorferi in the skin: a morphological and immunohistochemical study of the heterogenous appearance of this microorganismAberer, E., Kersten, et alB. burgdorferi granules were detected in skin biopsies of erythema migrans and acrodermatitis chronica atrophicans lesions.

Annals New York Academy of Sciences, 1988;539:468-470.

Concurrent neocortical borreliosis and Alzheimer's disease: Demonstration of a spirochetal cyst formMacDonald, A.B.In vivo finding of Borrelia burgdoreri cysts in an autopsy of a human brain. "An unexpected observation was the identification of cystic forms of the Borrelia spirochete in dark-field preparations of cultured hippocampus, and in imprints of hippocampus... A cystic form of the Borrelia spirochete would explain the ability of the microbe to persist in the host during a prolonged period of asymptomatic clinical latency, which spans the period between primary infection and the expression of tertiary manifestations of neuroborreliosis."

Borrelia - duttoniLancet, 1920 ;2:1237-1244.

(R) The Horace Dobell lecture on an experimental investigation of Spirochaeta duttoni, the parasite of tick feverLeishman, W.B.The author argues that S. duttoni, when inside a tick, reproduce by a process of budding and extrusion of granules; the granules grow into young spirochetes. The granules are also themselves capable of multiplication. Their development into spirochetal form within a vertibrate host is an exceptional occurence, brought about by certain environmental circumstances. This theory rests on an accumulation of observations by the author and by other researchers cited, including a correlation of the temporary disappearance of spirochetes from the tick's stomach with the appearance of large numbers of granules. Temperature was found to influence the development of classic-shaped spirochetes.

Annales de l'Institut Pasteur, 1918;32:49-59.

A note on the "granule clumps" found in Ornithodorus moubata and their relation to the spirochaetes of African relapsing fever (tick fever).Leishman, W.B.Granules developed into spirochetes. Innoculation of tissues containing only granules produced spirochaetosis in mice. Observed periods of several days where few, if any, spirochetes could be found inside a tick, followed by sudden re-invasion of tissues with mostly young and vigorously motile spirochetes; then this sequence would repeat in a regular pattern. Concludes that this has to do with the reproductive habits

Parasitology, 1912;Vol. IV, p.133.

The transmission of Spirochaeta duttoni.Hindle, E.[According to W. Burgdorferi, 1999:] Hindle reported that in infected ticks kept at 21SC, the spirochetes disappeared from the midgut by the 10th day and could no longer be detected either in the gut or in the tissues. However, triturates of such ticks were able to infect mice, and an increase in temperature to 35SC led to the reappearance of typical spirochetes.

Annals of Tropical Medicine and Parasitology, 1907;No. 3

The morphology and life-history of Spirochaeta DuttoniBreinl[According to Dutton, 1907:] Observed encysted forms of S. duttoni in the spleen. The cysts broke into granular bodies from which new generations of spirochetes emerged.

Lancet, 1907;ii:1523.

(R) A note on the morphology of Spirochaeta Duttoni.Dutton, J.S. & Todd,Spirochetes within sporocyst-like bodies were found in the blood even when other forms had disappeared. The authors suggest that reproduction may occur by direct division, but possibly also by a process involving extrusion of granules which subsequently develop into new spirochetes.

Borrelia - recurrentisCompt. rend. acad. sci., clix, 1914;pp. 119-122.(F) De la periode de latence du Spirille chez le Pou infecte de fievre recurrent.Sergent, E. & Foley,[As described by Leishman, 1920): After ingestion into ticks, the Leptospira studied disappeared after 24 hours. After the 6th day, new, actively mobile spirochetes reappeared suddenly in great numbers. Infectivity was highest on the 6th day prior to this reappearance, despite of the absence of demonstrable spirochetes. Transverse fission of the spirochetes in the louse was only rarely observed. When classic-shapedspirochetes were present, their numbers did not increase.

LeptospiraJournal of Bacteriology 1954;67:619-627.

(R) Formation of granular structures by Leptospirae as revealed by the electron microscope.Czekalowki, J.W., et alLeptospira began to show granulation after 2 weeks in a culture. The granules were spaced regularly within the bodies of the spirochetes. After four weeks a larger type of granule appeared which was broader than the body of the spirochetes. These were later "shed free." By the 5th to 7th month, there were no spirochetes observed; the culture contained only granules. The granules consisted of "what appears to be short segments of leptospiral body embedded in homogeneous substance." The authors conclude that the "formation of granules represents a rhythmic and constant process and hence these granules must play a role in the life-cycle of leptospirae."

J. Hygiene, 1949;47:390-392.

The morphology of the genus Leptospira as shown by the electron microscopy.Babudieri, B.

Acta Biologica Belge, 1943;3-4:245.

(F)Coloration des granules leptospiriens.Herreweghe, E.

Bull. ass. diploms microbiol. fac. pharm.,1942;61:72-80.

(F) Le micro-manipulateur et les granules d'une souche de Leptospire aquicole non-pathogene.Bessemans, A.,[According to Czekalowski, 1954]: Granules from the culture of a leptospira were isolated using a micromanipulator and grown from single cell

The etiology and mode of infection and specific therapy of Weil's disease.Inada, R., Ido, Y., et al,Interprets atypical spirochetal forms as "degenerative." Spirochetes were seen inside phagocytic cells and epithelial cells. Classic-shaped spirochetes were found in the blood in small numbers, and only in the early stages of the disease.

Non-SpirochetalJournal of Bacteriology, 1942;44:37-70.The significant of the large bodies and the development of L-type colonies in bacterial cultures.Dienes, L."If they [L type colonies] represent a variant form [of bacterial morphology], the observations indicate that the variability of bacteria extends much further than is commonly supposed. The bacteria apparently are able to grow in the form of soft granules and to multiply by the development and germination of large bodies." (p.60)

Other/Mixed/Unknown

CRC Press, 1993

Cell wall deficient forms: Stealth PathogensMattman, L.H.

Eur. J. Clin. Microbiol. Infect. Dis., 1989.

Unusual in vitro formation of cyst-like structures associated with human intestinal spirochaetosisGebbers, J.O. et alIn vitro findings suggest that spirochetes may develop in cysts, contrary to the traditional view that transverse fission is their main mode of reproduction. As cysts were found in centrifugates of cultures but not in biopsy speciments, the authors speculate that this mode of reproduction may occur only when in sub-optimal environments outside the host. Electron micrographs of maturation of spirochetes within the

(R) Acute plaques in multiple sclerosis, their pathogenic significance and the role of spirochaetes as etiological factor.Steiner, G. 1952

Journal of Bacteriology, 1951;62:347-349.

(P) Further studies on the significance of spirochetal granules.Hampp, E.G.

American Journal of Syphilis, 1950;34:122-125.

Studies on the life cycles of spirochaetes: I. The use of phase contrast microscopy.Delamater, E.D., et alIncludes several small photos of spirochetes emerging from "gemma," which the authors interpret as reproductive forms.

(R) "Silver cells" and "spirochete-like formations" in MS and other diseases of the central nervous systemHassin, G.B. et alReview confirms the findings of G. Steiner and other researchers who found "silver cells" [spirochetal granules that take a silver stain] in brain autopsies of MS cases. (G. Steiner contended that MS is an infectious disease caused by a spirochete that changes from a classic spirochetal form to a granular form, and which destroys myelin.) While the authors found granules in the CNS of all 8 MS patients they studied, they dispute Steiner's belief that they are the causative agent of the disease.

(R) Spirochaetes and their granular phase.Fantham, H.B.Observed the process of spirochetal formation of granules, and the emergence of small spirochetes from these granules. "It must also be borne in mind that coccoid bodies may be present when spirochaetes as such cannot be detected." (p.410)

(R) The infective granule in certain protozoal infections, as illustrated by the spirochaetosis of Sudanese fowl.Balfour, A.Spirochetes were observed to discharge large numbers of granules. "...the spirochaetes undergo an astonishing change. They discharge from their periplastic sheaths spherical granules, and it is apparently these granules which enter the red cells, develop in them and complete a cycle of schizogony...In process of time the spirochaete loses its activity, becomes difficult to see, and eventually all that is left of it is the limp and lifeless... [that the granules] do not appear to take on the Romanowsky stain may explain why they have not previously been noticed... I have found these granules to be resistant forms and their presence in countless numbers in the tissues might explain part of the mechanism of relapse and the difficulty of curing completely some of the more chronic spirochaetal infections, as, for example, syphilis and

Journal of Infectious Diseases, 1906;3:291-293.

Studies on the Spirillum obermeieri and related organisms.Novy, F.G., & Knapp,Found that the classic spiral form is not the only form that spirochetes may assume.

Treponema ...Zbl. Bakt., 1994;280:297-303.

Formation of multiple treponemes.Wolf, V. & Wecke, J.[From the abstract:] "It was calculated that the formation of spherical bodies may reduce their surface by up to 75% as compared to the single form. Thus, the reaction surface for antibodies or other compounds produced by the host is considerably diminished. Therefore, such spherical structures being at resting states may represent a survival strategy of spirochetes. ...the spherical bodies may be the starting point of the new inflammatory episode. This wavelike process is typical of many spirochetal diseases."

Vestn Dermatol Venerol, 199?;4:32-6.

(F)The cytoarchitectonics of hard chancre in rabbits with experimental syphilis exposed to oliusulfon and cefamezine[In Russian; English abstract available]Delektorskii VV.,Describes T. pallidum ultrastructure, and the process of formation of a granule. Cefamezin did not effect spirochetal cysts in the treatment of (snip)

Microbiol. Immunolo.,;1984;28:11-22.

Colonial morphology of treponemes observed by electron microscopy.Umemoto, T., et al,[From the abstract:] "Scanning and transmission electron microscopy revealed that the colonies of Reiter treponemes contained spherical forms almost up to 5 am in diameter, each consisting of an outer membrane and a treponemal main body."

Journal of Applied Bacteriology, 1983;55:417-428.

(R)A proposed life cycle for the Reiter treponeme.Al-Qudah, A.A.Demonstrates the viability of cysts and the existence of a complex manner of reproduction. "Although transverse fission may be the main mode of reproduction of Reiter treponemes in optimal growth conditions, the spontaneous formation of cysts increases in aging cultures to the extent that it is rare to find a typical treponeme in old cultures. We conclude that such cysts... [serve to] by-pass adverse environmental conditions and to ensure the propagation of the organism. ...the existence of the causative agent of syphilis in a nonspirochetal form has long been hypothesized to explain the latency of syphilis and the infectivity of tissues devoid of demonstrable treponemes...This agrees with what usually happens in protozoa in nature; ...the majority of cysts in protozoa are a means of protecting their contents against unfavorable conditions... Later, depending on conditions when the harmful exposure is past, protective cysts may become multiplication cysts. They are not

Microbiol. Immunolo. ,1982;26(3):191-198.

An internal view of the spherical body of Treponema macrodentium as revealed by scanning electron microscopy.Umemoto, T., et al,"External observation of a spherical body by scanning electron microscopy clearly revealed the main bodies [spirochetes] running beneath the inner surface of the spherical body membrane [cyst]. " Includes a freeze fracture photograph of a cross-section of a multispirochetal cyst,

Acta Pathol Microbiol Scand [A],1977;pertenue (sic KMD) Electron microscopy of lymph nodes of hamsters experimentally infected with TreponemaBlom J.Treponemes were found intracellularly in macrophages. These treponemes did not show their typically helical shape, but were present as spherical forms or cysts.

J. Am. Vener. Dis. Assoc. , 1976;3(2):109-127.

Biopharmacology of syphilotherapy.Rein, M.F.

British Journal of Venereal Diseases, 1971

(P) Current concepts of the morphology and biology of Treponema pallidum (syphilis) based on electron microscopyOvcinnikov, N.M., et al[Granules:] "Another mode of reproduction resorted to in adverse circumstances consists in the formation of spores which subsequently develop into new treponemes. The breakdown into granules is especially pronounced under the action of penicillin and immune sera." [Cysts:] "Under stressful conditions, the treponeme 'packs' itself into a compact roll (Fig. 8) and becomes covered with a transparent mucoid capsule, which resists the pentration of drugs and antibodies." "Encystment as a mechanism of survival and mode of reproduction is widespread in nature, especially among protozoa." [Intracellular:] T. pallidum were found inside a cell taken from the site of a chancre; and L-forms were found

New England Journal of Medicine, 1971; 284: 642-653.

Diagnosis and treatment of syphilis.Sparling, P.F.Includes a review of recent [as of 1971] evidence indicating that penicillin treatment is not always curative in patients with late syphilis. "Penicillin therapy of neurosyphilis has not been as effective [as in early syphilis]. Several studies have reported relapses... Clinical progression of symptomatic neurosyphilis is relatively common despite antibiotics." (p.650) [Diagnostic issues:] Some infected patients also

British Journal of Venereal Diseases, 1968;44:1-34.

(P) Further study of ultrathin sections of Treponema pallidum under the electron microscrope.Ovcinnikov, N.M., et al,Observations of T.pallidum cystic and granular formations under the electron microscope. "...under unfavourable conditions of existence, treponemes form real cysts as a method of persistent survival and multiplication, as occurs not infrequently among protozoa." "As the treponeme moves, the thickness changes. This indicates that the body possesses a capacity for contraction... The sharply-marked structural elements of the treponeme and its complex and characteristic structure indicate that cysts are not a product of degeneration. In addition, in cultures where there are many cysts, they are very mobile, which is another argument against degeneration... When transfers are made from cultures containing cysts and almost no ordinary spiral forms, growth of ordinary spiral forms occurs." Includes photo of a treponeme packed into a cyst

British Journal of Venereal Diseases, 1968

Further observations on the persistence of Treponema pallidum after treatment in rabbits and humans.Yobs, A.R.Results of a 4-year study of rabbits treated with penicillin for late latent syphilis. Confirmed persistence of syphilis in numerous subjects after antibiotic treatment. Cortisone treatment can reactivate clinical disease. Offers various theories to explain the persistence of T. pallidum, including morphologic changes in the organism. However, the author believes that the existence of a complex life cycle with differing

Significance of spiral organisms found after treatment in late human and experimental syphilis.Collart, P., Borel, et alPersistence of T. Pallidum after treatment. Organisms are still present but have lost their virulence. Cortisone reactivates clinical disease.

Journal of Bacteriology, 1963;85:932-939.

Morphology of Treponema microdentium as revealed by electron microscopy of ultrathin sectionsListgarten, M.A., et al,[Findings:] Spirochetal granules were found in cultures of T. microdentium. They were more numerous in older cultures. [Observations pertaining to the classification of spirochetes:] "The [cell] envelope had an irregular contour, was easily disrupted during processing, and did not appear esstial in maintaining the shape of the protoplasmic cylinder. It is therefore probable that this envelope is quite distinct from bacterial cells walls, which in ultrathin sections appear as regular, well-defined, electron-dense structures." (p.938)

Journal of Bacteriology, 1961;82:967-978.

Influence of osmotic pressure on the morphology of the Reiter treponeme.Hardy, P.H. & Nell"[Reiter] Treponemes in saline solution were observed while distilled water was pulled into the preparation by capillary action, and it was found that although all treponemes in a field were not changed to spheres simultaneously, the conversion of any single one took place

American Journal of Syphilis, 1953;37:29-36.

Treponema pallidum buds, granules and cysts as found in human syphilitic chancres and seen in fixed unstained smears under darkground illumination.Coutts, W.E. et al"Spirochetogenic granules are by far more numerous than the cysts."

American Journal of Syphilis, 1951;35:164-179.

Studies on the life cycles of spirochaetes: V. The life cycle of the Nichols non-pathogenic Treponema in culture.Delamater, E.D., et alFormation of reproductive cysts.

American Journal of Syphilis, 1951;35:216-224.

Studies on the life cycles of spirochaetes: VII. The life cycle of the Kazan non-pathogenic Treponema pallidum in culture.Delamater, E.D., et al

Yale Journal of Biology and Medicine, 1950.

The morphology and staining characteristics of Treponema pallidum. Review of the literature and description of a new technique for staining the organisms in tissues.Campbell, R.E. et al

Journal of Experimental Medicine, 1950;92:247-250.

(P) Studies on the life cycles of spirochetes: IV. The life-cycle of the Nichols pathogenic Treponema pallidum in the rabbit testis as visualized by means of stained smears.Delamater, E.D., et al

Experimental Medicine, 1950;92:239-246.

Studies on the life cycles of spirochaetes: III. The life cycle of the Nichols pathogenic Journal of Treponema pallidum in the rabbit testis as seen by phase contrast microscopy.Delamater, E.D., et al"...it seems likely from these observations that there are two means of vegetative reproduction, consisting of (1) transverse division (the most important under usual conditions); and (2) the production of gemmae or buds which eventuate into unispirochetal cysts comparable to those described for saprophytic forms, within each of which single spirochetes develop and differentiate, and from which they subsequently emerge."

American Journal of Syphilis, 1949;33:101-113.

Morphology, cultural characteristics and a method for mass cultivation of the Reiter spirochaetes.Gelperin, A.

Journal of Bacteriology, 1948;56:755-769.

(P) Morphologic characteristics of certain cultured strains of oral spirochetes and Treponema pallidum as revealed by the electron microscope.Hampp, E.G., Scott"Typical free granules, the end products of granule "shedding," ... consist for the most part of what appear to be short sections of spirochetes closely packed together...Although it is not possible to determine from these micrographs that the granules are germinative units, their constant rhythmic occurrence in living cultures suggests this possibility. Further support of this hypothesis is provided by the fact that cultures up to 31 months old, showing only refractile granules by dark-field examination, have invariably given normal growths on transfer to fresh medium (Hampp, 1946)." (p.768). Also of interest: the authors did not find cell membranes on the spirochetes they examined.

Bulletin of Hygiene, 1947;23:548.

Study by means of micromanipulation of the virulence of one or several spirochaetes as well as viability of spirochaetes or granular forms of culture of supposed TreponemaBessemans, A.,

American Journal of Syphilis, 1947;31:109-114.

Transmission of experimental syphilis from mouse to mouse in absence of S. pallida and pathologic changes in presence of successful innoculationWile, U.J.Showed that syphilis can be transmitted by tissues from infected hosts in the absence of spirochetes, suggesting that the infectious agent is present in another form. Note: this study does not specifically mention cysts or granules.

J Am Dental Assoc, 1946;33:201-206.

Morphologic alteration of smaller oral treponemas during aging of cultures; Effect of age on viability of spirochetal cultures.Hampp, E.G.

Journal of Bacteriology, 1943;46:15-24.

(P) Bacterial morphology as shown by the electron microscope; V. Treponema pallidum, Treponema macrodentium and Treponema microdentium.Mudd, S., Polevitsky, et al"Irregularly spheroidal, dense bodies... are often found attached to the spirochetal cell, frequently near the end; such a dense body may be in close apposition to the outside of the spirochetal cell-wall or may be connected to it by a short stalk. The evidence concerning these bodies seems to support the interpretation that they are asexual reproductive bodies." (p.23)

American Journal of Syphilis, 1942;26:565-573.

Some morphologic features of the Nichols strain of Treponema pallidum as revealed by the electron microscopeMorton, H.E. et al,

JAMA, 1942;199:880-881.

(P) The morphology of spirochaeta pallida in the electron microscope.Wile, U.J., Picard, et al"...in many specimens a curious knoblike structure was seen at the end of many organisms. Their almost uniform shape and density suggest that these are not extraneous particles of the preparation but a part of the organism itself."

(R) The life history of Treponema pallidum. A Critical review of literature.Ingraham, N.R., Jr.Excellent, "must-read" review of the findings and various interpretations of T. pallidum prior to 1932. States that there have been 18 separate experiments in which tissues from infected hosts transmitted infection in the absence of spirochetes, suggesting that the organism is present in another form. Of particular interest to the reader: Roukavischnikilff's belief that the cause of syphilis circulates in the blood of an infected animal in an avisual (submicroscopic) stage; and McDonaugh's theory that Treponema pallidum is the adult male phase of a coccidial

(P) Granular transformation of Spirochaeta pallida in aortic focal lesions.Warthin, A.S. et alFindings of T. pallidum spirochetes in atypical forms in aortic focal lesions suggested that the possibility that the spirochete may transform itself into a minute granule by a series of contractions. Atypical forms were found even when typical spirochetes were absent. Includes an interesting drawing of the transitional stages observed as a spirochete transforms itself into a minute granular form. The authors raise the question as to whether this progression represents evolution or involution, but seem to emphasize the possibility of involution.

British Medical Journal of Dermatology & Syphilis, 1913;25:1-14.

(R) The complete life history of the organism of syphilisMcDonagh, J.E.R.

Lancet, 1912;2:1011.

The life cycle of the organism of syphilis.McDonagh, J.E.R.Poses several questions: (1) Why is the incubation period of syphilis so long? (2) Why do 1-2 injections of salvarsan [used to treat syphilis prior to the discovery of penicillin] not cure every case? (3) In tertiary syphilis, fewer organisms are found; why then is this stage the hardest to treat? The author argues that the Treponema pallidum is the adult male phase of a coccidial protozoan, and that the spores that result from the conjugation of the two sexual phases are the actual infectious agent of syphilis. The spores were observed to develop inside of

Journal of Experimental Medicine, 1912;16:194-198.

Treponema mucosum (new species) a mucin producing spirochaeta from pyorrhea.Noguchi, H.Irregular spirochetal forms and many granules appeared when conditions were unfavorable to the organism. Small spirochaeta were seen attached to round bodies as if they had just sprouted from them. Journal of Experimental Medicine, 1911;XIV:99-112.

A method for the pure cultivation of pathogenic Treponema pallidum.Noguchi, H.Observed "spore-like round bodies" connected with young pallida. Also of interest: the author reports that he was unable to cultivate T. pallidum in any medium without the addition of tissue.

Journal of the Royal Army Medical Corps., 1911;Vol. XViI, p.225.

Granule-shedding in Treponema pallidum and associated Spirochaetae.O'Farrel, W.R. Balfour, A

Münchner med. Wochenschr., 1906;53:310-312.

(F) Weitere Mitteilungen über die Spirochaeta Pallida.Herxheimer, K.Found that the classic spiral form is not the only form that spirochetes may assume.

Gemmae -size from 7 microns to 30 microns { These are the largest round bodies of spirochetes - also called Cystic borrelia}

Granules - size from0.5 microns to 2 microns {these are the agents of Granulovacuolar Degeneration of neural cells in Alzheimer's Disease. Granular spirochetal forms are very prominent inside of Biofilms of Borrelia where they co-exist with other non-spiral specialized borrelia forms. Eisendle's images of ACA lesions with FFM technique nicely demonstrate aggregates of pure granular borrelia [ which I now reclassify as biofilm communities of Bb in ACA skin. As to the maximum number of Granules which a single spirochete can produce, I would place an upper limit of between 30 to 50 granules Maximum per single spirochete.

Liposomes - size from 0.01 microns to 0.03microns. Liposomes are continually shed from the surface of the spiral motile borrelia as they move through the body. There seems to be no limit to the number of liposomes which can be shed from a single borrelia spirochete. [Liposomes = "Blebs" = Microvescles] The continuous shedding of liposomes [ without numerical upper limits on the number of liposomes emerging from a single spirochete is a biological Multiplier of pathogenicity, vastly expanding the damage that a single spirochete can cause in its lifetime.

All contain DNA ,ButGemmae - DNa content is the entire chromosome and all 21 plasmidsgranules - DNa content is the entire chromosome and all 21 plasmidsLiposomes - DNa content is probably limited to Plasmids only ( linear and circular - Garon's studies)

the Beermann paper from year 2000 clearly shows the profile of liposomes (blebs)under electron microscopy.Radolf's paper in year 2012 shows blebs to be much smaller than gemmae ( cystic borrelias)

liposomes can easily invade nonphagocytic mammalian cells [ Beermann 2000]liposomes [blebs] are photographed inside the host mammalian cell nucleus.Gemmae are incapable of doing this , due to their size.

Granules can also form inside mammalian cells, either as a a result ofpenetration of the cell by the motile spiral form and then subsequentsegmentation breakup into granular formsorperhaps granular forms can directly penetrate mammalian cellsto reach the cytoplasm, [ but granular forms are probably incapable ofpenetrating the nucleus of the mammalian cell- at least based on imagedata now available]

1. is there eve a circulating cystic borrelia form i human blood? answer: Yes - I can send to you a 200 slide lecture on Borrelia Cystic forms if you wish to see it. It is also freely availabe on my websitewww.alzheimerborreliosis.net2. Can cysts of borrelia be demonstrated in direct analysis of human blood? answer --Yes -- see link to Round body (Cystic borrelia lecture-200 slides) - - DR Morten-Laane3. Is the content of Cystic borrelia equal to the DNA content of spiral borrelia? answer --yes4. Is a culture negative result ( of blood ) the end of the story as far as the possibility that cystic forms might be present in tissue sites outside of blood? answer -- Negative in blood does not exclude the existence of cystic forms elsewhere. 5. Does any lab testing method reflect the Status of :SANCTUARY SITES [ BRAIN,EYE,GONAD,FASCIA ETC] ?? Answer -- No Lab test other than the AUTOPSY Is informative about the status of SANCTUARY SITES in the human body for the possible presence of any of the forms of borrelia ( Spiral, Straightened, Cystic,Granular, Cell Wall deficient, Biofilm communities,Liposomal forms of Borrelia)